The cell biology of protein folding in the lumen of the ER of eukaryotic cells will be analyzed by genetic, biochemical, immunochemical and molecular methods. The folding and oligomeric assembly of proteins in this highly specialized compartment are complex processes involving a number of co- and posttranslational modifications and interactions with numerous molecular chaperones and folding enzymes, and a system of quality control for the sorting of correctly folded and incompletely folded products. Detailed studies are proposed using viral membrane glycoproteins and some cellular proteins as models in live tissue culture cells, in Saccharomyces cerevisae, and, after in vitro translation, in isolated microsomes. Our aims are; 1) To evaluate the role played by the milieu and by individual ER chaperones in the folding process. 2) To investigate the folding and trimerization pathway taken by influenza hemagglutinin (HA), a well-characterized model protein. 3) To investigate folding events that occur cotranslationally in the ER with particular emphasis on interactions between the growing nascent chains and the translocon- associated proteins and chaperones. 4) To identify and characterize new folding and quality control factors. A targeted search will be performed using folding-incompetent mutant versions of bovine trypsin inhibitor, RNase A, Staphylococcus nuclease and influenza HA. A screen for quality control mutants in S. cerevisiae will be developed, and the mutants analyzed by genetic and biochemical methods. 6) To develop methods for identifying folding domains and secretion-competent fragments of secretory and membrane proteins produced in the ER. The results will expand our understanding of the ER as a protein folding compartment, and provide new insights in the molecular and cell biological basis for the post-translational quality control systems that determine the fidelity of protein expression. The results will also be relevant for a variety of human diseases with an ER-storage disease phenotype, and for biotechnology as it addresses problems in the expression of recombinant proteins in heterologous cell types.